We typically use 0.5ug of E. coli genomic DNA in a labeling reaction for each hybridization. The genomic DNA was fragmented to 500 to 1000bps before labeling. The following protocol should produce enough labeled probe for 8 hybridizations.
For labeling 4ug Genomic DNA:
| Genomic DNA |
1.9ug/ul |
2.1ul |
| Random Hexamer |
5mg/ml |
1ul |
| H2O |
|
14.9 |
|
| Total |
|
20ul |
Heat to 95C for 5min, place on ice for 5min
Labeling
| DNA Mix |
|
20ul |
| dAGC |
5mM each |
5ul |
| EcoPol Buffer |
10x |
5ul |
| CyDye-dUTP |
1mM |
2ul |
| H2O |
|
17ul |
| Klenow Fragment |
50u/ul |
1ul |
|
| Total |
|
20ul |
Incubate at 37°C for 3.5 hours
Add 2.5ul 0.5M EDTA to stop reaction
Clean up Labeled Probes
-
Prewash Microcon-30 microfilter by adding 450ml miliQ H2O and spinning for 10 min. @ 12,000 RPM.
-
Add 450ml miliQ H2O to each of the probe samples (or total 500ul). Mix thoroughly by pipetting up and down. Transfer samples to separate Microcon-30 microfilters. (Amicon)
-
Spin at 12,000 RPM in microfuge for 10 minutes or until 20-40 ml remains in the filter.
-
Add 450 ml miliQ H2O to the probe and gently mix by pipetting up and down. Be careful not to touch the filter at the bottom of the filtration unit.
-
Spin 10 minutes at 12,000 RPM.
-
Repeat step 4 , spin 12min to get smaller volume.
-
Invert column into a fresh tube and spin 1 minute at maximum speed to recover probe. Carefully measure recovered probe volume if necessary.
Probe can be stored at 4°C or -20°C in dark for further use.
Reagents and Suppliers
| Cy3-dUTP |
1mM |
Perkinelmer |
NEL578 |
| Cy5-dUTP |
1mM |
Perkinelmer |
NEL579 |
| Klenow Fragment |
50U/ul |
NEB |
M0210M |
| 100 mM dNTP set* |
10X |
Amersham |
27-2035-01 |
| pd(N)6 Sodium Salt (Hexamer) |
50U |
Amersham |
27-2166-01 |
| Microcon YM-30 column |
|
Amicon |
42410 |
*for 10X stock: 5 mM each of dA, dG, dC.
|
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